Progressive cerebral deposition of the amyloid beta-protein (Abeta) is an early and invariant feature of Alzheimer's disease (AD) which appears to precede dementia by many years. In at least a few families, betaAPP mismetabolism and Abeta deposition are believed to represent the molecular basis of the disease. Recently, Abeta was discovered to be continuously produced and secreted as a soluble peptide by cultured cells during normal metabolism. As a result, the mechanism of Abeta production can now be studied dynamically in vitro and the effects of pharmacological modulation readily assessed. Here, we propose an integrated series of molecular and cell biological experiments which build on this discovery by examining the physiological control of Abeta production. Our Specific Aims are: 1) To examine the mechanism and regulation (Abeta secretion using both cDNA- transfected human cell liens and primary human donor cells (neurons, fibroblasts, endothelial cells). We will search for cell-type specific differences, effects of donor age, effects of basal and activated betaAPP phosphorylation state, and effects of several cellular stressors (serum deprivation; excess calcium; oxidative injury; etc.) on the production, release and stability of Abeta. 2) To establish the structural requirements for cellular generation of Abeta from betaAPP using site- directed mutagenesis around the N- and C-termini of Abeta as well as deletions of specific functional domains or consensus sequences within betaAPP [e.g., NPXY internalization signal; KPI domain; growth promoting motifs (e.g., RERMS); acidic domain]. Some of these mutated betaAPP constructs should provide critical substrates for the confirmation or exclusion of any candidate proteolytic enzymes which putatively release the Abeta fragment. 3) To define the molecular mechanisms of increased Abeta accumulation in various genetic forms of FAD, both those on chromosome 21 (betaAPP missense mutations) and those on chromosome 14, using primary donor cells (fibroblasts). This approach should provide information about betaAPP metabolism and Abeta production in endogenous cells from chromosome 14 donors even before the defective gene is identified. 40 To search for evidence of polarized secretion and/or transcytotic transport of Abeta in epithelial and endothelial monolayers, mimicking the microvessel wall. This approach seeks to provide experimental evidence for the longstanding but untested hypothesis that beta-amyloid, particularly in blood vessels, could arise from a vascular or circulating source. Taken together, these experiments address central questions about the mechanism of Abeta generation, its alteration in familial Alzheimer's disease and its regulation by a variety of physiological and pharmacological modulators. As such, this research should provide insights not only into the normal metabolism of betaAPP and Abeta but also about how one might control Abeta production by a variety of pharmacological strategies. The work addresses one of the most promising pathogenetic and therapeutic hypotheses of Alzheimer's disease.